The nucleosome is the building block of chromatin, the condensed form of DMA found in the cell nucleus. First, a tetramer of histones H3 and H4 binds to DNA to form the tetrasome, which then binds two H2A-H2B dinners to form the nucleosome. This structure is further compacted by the linker histone (H1) that binds the linker DNA, further compacting the DNA and limiting protein-DNA interactions that are necessary for gene expression. Regulation of chromatin dynamics is critical for cell survival. Herein I propose to investigate two ATP-independent ways in which the cell regulates chromatin dynamics and thereby DNA accessibility: the incorporation of histone variants into nucleosomes and the role of histone chaperones in DNA accessibility. Specific histone dimer variants are often found near locations of active transcription (gene expression);therefore, histone variants are thought to be more dynamic thereby facilitating efficient DNA-protein interactions. Furthermore, histone dimer variants may form less compact, or partial, nucleosomes. These partial nucleosomes (or hexasomes) have been observed as intermediates during transcription. Altering the free concentration of dimers could facilitate the formation of these partial nucleosomes. Therefore, a second method for the cell to regulate chromatin dynamics is through the use of histone chaperones to control the free histone concentration. I propose to use Nucleosome Assembly Protein 1 (NAP1) as a means to control free histone concentrations, to measure the affinity and cooperativity of histone dimers and variants, and to probe DNA accessibility to proteins. These studies will aid our understanding of the differences in chromatin dynamics between nucleosomes containing major type and variant dimer histones, including examination of these properties under sub-saturating conditions, which may exist during transcription. Additionally, I will probe the roles of the linker histone H1 and NAP1 in chromatin dynamics, and define the NAP1-H1 interaction using X-ray crystallography. The results from these studies will enhance our knowledge of how proteins gain access to nucleosomal DNA and how cells regulate this by controlling free histone concentrations. These data should also aid in our understanding of transcription in the context of chromatin.